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Technical Refolding of Proteins: Do We Have Freedom to Operate? Maria Eiberle, Alois Jungbauer

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Technical Refolding of Proteins: Do We Have Freedom to Operate? Maria Eiberle, Alois Jungbauer Technical refolding of proteins: Do we have freedom to operate? Maria Eiberle, Alois Jungbauer To cite this version: Maria Eiberle, Alois Jungbauer. Technical refolding of proteins: Do we have freedom to operate?. Biotechnology Journal, Wiley-VCH Verlag, 2010, 5 (6), pp.547. 10.1002/biot.201000001. hal- 00552343 HAL Id: hal-00552343 https://hal.archives-ouvertes.fr/hal-00552343 Submitted on 6 Jan 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Biotechnology Journal Technical refolding of proteins: Do we have freedom to operate? For Peer Review Journal: Biotechnology Journal Manuscript ID: biot.201000001.R1 Wiley - Manuscript type: Review Date Submitted by the 30-Mar-2010 Author: Complete List of Authors: Eiberle, Maria; Boehringer Ingelheim RCV Jungbauer, Alois; University of Natural Resources and Applied Life Sciences, Department of Biotechnology Primary Keywords: Biochemical Engineering Secondary Keywords: Bioseparation Keywords: refolding, patent, continuous Wiley-VCH Page 1 of 28 Biotechnology Journal 1 For submission to Biotechnology Journal Ms. No. biot.201000001 2 3 4 5 6 7 8 9 10 11 12 Technical Refolding of Proteins, Do we have Freedom to 13 14 15 Operate? 16 1,3 2* 17 Maria K. Eiberle , Alois Jungbauer 18 1 19 Boehringer Ingelheim RCV & Co GmbH, Vienna, Austria 20 2 Department of Biotechnology,For University Peer of Natural ReviewResources and Applied Life Science, Vienna, Austria 21 22 3 current address Rentschler Biotechnologie GmbH, Laupheim, Germany 23 24 25 26 st 27 1 revision 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 * Corresponding Author: 58 59 Mailing address: Muthgasse 18, 1190 Wien, Austria 60 E-mail address: [email protected] Tel.: +43 136006 6226; Fax: +43 1 3697615 1 Wiley-VCH Biotechnology Journal Page 2 of 28 1 Abstract 2 3 4 Expression as inclusion bodies in Escherichia coli is a widely used method for the large-scale 5 production of therapeutic proteins, that do not require post-translational modifications. High 6 7 expression yields and simple recovery steps of inclusion bodies from the host cells are 8 9 attractive features in the industrial scale. However, the value of an inclusion body based 10 11 process is dominated by the solubilization and refolding technologies. Scale-invariant 12 13 technologies, economically and applicable for a wide range of proteins are requested by 14 industry. The main challenge is to convert the denatured protein in its native conformation at 15 16 high yields. Refolding competes with misfolding and aggregation. Thus, yield of native 17 18 monomer depends strongly on the initial protein concentrations in the refolding solution. 19 20 Reasonable yields areFor attained atPeer low concentrations Review ( ≤ 0.1 mg/mL). However, that requires 21 large buffer tanks and time-consuming concentration steps. We attempt to give an answer to 22 23 which extent refolding of proteins is protected by patents. Low-molecular mass additives have 24 25 been developed to improve refolding yields through the stabilization of the protein in the 26 27 solution and shielding hydrophobic patches. Progresses were established in the field of high- 28 pressure renaturation and on-column refolding. Mixing times of the denatured protein in the 29 30 refolding buffer were reduced by newly developed devices and the introduction of specific 31 32 mixers. Concepts of continuous refolding were introduced in order to reduce tank sizes and 33 34 increase yields. A few patents covering refolding of proteins will expire soon or have expired. 35 36 This gives more freedom to operate. 37 38 39 40 41 Keywords: Inclusion bodies, refolding, E. coli , recombinant proteins, on column, additives 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 Wiley-VCH Page 3 of 28 Biotechnology Journal 1 2 3 Introduction 4 5 6 Approximately 40 % of all biopharmaceuticals are produced in E. coli cells [1]. E. coli cells 7 grow rapidly to high cell densities on inexpensive substrates and well established 8 9 fermentation strategies are attractive for an economic expression in industrial scales. 10 11 Furthermore, the genetic properties of E. coli are well characterized and the strains are easy to 12 13 handle. This explains why E. coli is an economic and efficient production system, and widely 14 15 used for the expression of recombinant proteins [2,3]. However, recombinant proteins are not 16 always folded in their proper and active conformation during protein biosynthesis. For a broad 17 18 majority of heterologous proteins, secretion in E. coli results in 0.5 to 0.8 g L-1 volumetric 19 20 yield [4]. Higher titersFor can be attained,Peer but usually Review require an extensive engineering of the 21 22 fermentation protocol and expression system [5,6,7]. Thus many products on the market are 23 produced as inclusion bodies in the cytoplasm of E. coli , where high fermentation titers can be 24 25 achieved according to Biopharmaceutical Products in the US and European Markets 6th 26 27 edition. Inclusion bodies contain the target protein as insoluble aggregates, present in a kind 28 29 of paracrystalline form. The proteins exist in non-native conformations but with a certain 30 31 content of secondary structure elements [8]. After solubilization with chaotropic buffers in a 32 reducing environment an elaborative renaturation step is required to refold the protein in its 33 34 native and active conformation [9]. Anyway, this technology is widely used. Beside high 35 36 expression yields there are other benefits, that compensate the disadvantage of an additional 37 38 refolding step: Inclusion bodies have a higher density (~1.3 g/mL) than other cellular 39 components and cell debris and can be easily separated and purified by a combination of cell 40 41 homogenization and centrifugation. During expression the target protein accumulates in the 42 43 inclusion bodies and is mostly resistant to proteolytic attacks of cell proteases. After primary 44 45 isolation of inclusion bodies adhesive impurities such as cell debris and host cell proteins can 46 47 be reduced by several wash and centrifugation steps. Finally a high purity of up to 90 % of the 48 product protein can be achieved in inclusion bodies. This simplifies and reduces subsequent 49 50 purification steps [10,11]. 51 52 The process for the production of recombinant protein from inclusion bodies comprises cell 53 54 cultivation and harvest, disruption, recovery of inclusion bodies, solubilization, refolding and 55 reoxidation of disulphide bonds and further purification steps (Figure 1) [11,12]. If inclusion 56 57 bodies contain high amounts of impurities, a denatured purification step of the solubilized 58 59 protein can be performed prior to refolding. Popular methods are ion exchange, size exclusion 60 or metal ion affinity chromatography [13,14,15]. 3 Wiley-VCH Biotechnology Journal Page 4 of 28 1 Inclusion bodies are aggregated and densely packed paracrystalline forms of protein. These 2 3 refractile particles are solubilized in high concentrations of chaotropic agents such as urea or 4 guanidine hydrochloride. Reducing conditions are inevitable, as non-native intra- and 5 6 intermolecular disulfide bonds may have been formed in inclusion bodies during translation. 7 8 Solubilization results in a protein in its denatured form. The subsequent step transfers the 9 10 unfolded and reduced protein into conditions, where the formation of native and bioactive 11 12 structures is favored. Of all process steps, refolding is the most crucial step. It decides on the 13 efficiency of an inclusion body based process [16,17,18]. 14 15 Renaturation is initiated by reducing or removing the chaotropic solvent. The yield of the 16 17 refolding step depends strongly on the renaturation conditions such as pH, redox conditions, 18 19 buffer additives and protein concentrations. These conditions have been empirically optimized 20 for each individual protein.For Peer Review 21 22 Most proteins contain cysteine residues that form disulfide bonds, which are required for the 23 24 formation of a native, bioactive structure. For in vitro refolding it is usually essential to add a 25 26 redox buffer system to support the formation of native disulfide bonds. Supplementing the 27 28 refolding buffer with low molecular weight thiol reagents allows the formation of disulfide 29 bonds, as well as the reshuffling of incorrect formed bonds. Generally a combination of a 30 31 reduced and oxidized component is used, for example cysteine/cystine or reduced/oxidized 32 33 glutathione. Suitable ratios must be found to maximize yields [16,19]. Molar ratios of reduced 34 35 to oxidized agents are recommended between 5:1 and 1:1. These ratios provide a suitable 36 redox potential for the formation and reshuffling of disulfide bonds [20,21]. 37 38 However, the correct folding pathway competes often in disadvantage, with aggregation and 39 40 misfolding of the target protein. These two reactions dominate the efficiency of the in vitro 41 42 refolding step. After dilution of the unfolded and reduced protein in a refolding buffer, 43 transient intermediates (I) are formed (Figure 2). Usually, these intermediates are partially 44 45 folded and hydrophobic patches are not completely buried in the core of the protein.
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